The present invention relates to the field of quantitative microspectroscopy, and in particular to a method for calibrating a sample analyzer which is preferably a disposable sample analyzer.
The determination of such blood parameters as the Hematocrit (xe2x80x9cHCTxe2x80x9d), the Volume of single Red Blood Cells (xe2x80x9cRCVxe2x80x9d), the Mean Cell Volume (xe2x80x9cMCVxe2x80x9d) and the Red Cell Distribution Width (xe2x80x9cRDWxe2x80x9d) are of eminent clinical interest. Usually, systems based on electrical impedance measurement (Coulter Counter) or based on light scattering (Flow Cytometer) are employed (see. e.g., J. B. Henry, xe2x80x9cClinical diagnosis and management by laboratory methodsxe2x80x9d, W. B. Saunders Company, Philadelphia, 1996, pp. 548 ff. or D. H. Tycko, M. H. Metz, E. A. Epstein, A. Grinbaum, xe2x80x9cFlow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentrationxe2x80x9d, Applied Optics 24 (1985), 1355-1365). Impedance counters are complex and expensive instruments that require very careful adjustment and control of instrument and sample parameters. A major disadvantage of flow cytometers is the fact that the parameters of light scattering depend not only on cell volume, but also on the cell""s shape.
In 1983, Gray, Hoffman and Hansen proposed a new optical method for determining the volume of cells in a flow cytometer (M. L. Gray, R. A. Hoffman, W. P. Hansen, xe2x80x9cA new method for cell volume measurement based on volume exclusion of a fluorescent dyexe2x80x9d, Cytometry 3 (1983), 428-432). In this method, the cells are suspended in a fluorescent dye, which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence intensity which is directly proportional to the cell""s volume. In a flow cytometer, a single cell is passing through the laser-illuminated spot within approximately 10 xe2x96xa1s. Due to this short data acquisition time interval, the electronic detection bandwidth has to be relatively large, which results in a poor signal-to-noise ratio and in a low precision for the volume determination.
The available data acquisition time can be significantly increased by suspending the cells in a stationary sample and applying digital imaging fluorescence microscopy (see P. L. Becker, F. S. Fay, xe2x80x9cCell-volume measurement using the digital imaging fluorescence microscopexe2x80x9d, Biophysical Journal 49 (1986), A465). In the digital fluorescence microscopy approach, a calibration procedure is required in order to determine the cell volume. Recktenwald and co-workers have introduced a method where the calibration is performed by means of optical transparent and non-fluorescent microspheres that are suspended together with the cells (D. Recktenwald, J. Phi-Wilson, B. Verwer, xe2x80x9cFluorescence quantitation using digital microscopyxe2x80x9d, Journal Physical Chemistry 97 (1993), 2868-2870). The volume of individual spheres is determined by measuring their projection area under the microscope and transforming this number into a volume, assuming an ideal spherical shape. The decrease in fluorescence intensity as a result of the spheres"" volume that is being excluded from emitting fluorescence is used as the required calibration parameter. The advantage of this approach is given by the fact that the calibrating particles are located within the sample itself. In other words, a calibration is performed on the very same sample container, and no extra calibration sample is required.
The use of calibration spheres within a cell suspension is not without problems. First, the introduction of the spheres represents an additional step in the workflow. In systems that are designed for high throughput, this additional step would represent a disadvantage. Secondly, Recktenwald and co-workers observed a tendency of the fluorescent dye molecules to settle down on the sphere""s surface, which causes an error. Third, if the optical index of refraction of the spheres does not match well with the liquid""s index, then refraction-based artifacts in the measured fluorescence intensity occur at the edges of the spheres. And, finally, the use of microspheres can represent a problem, if e.g. a thin sample thickness in the order of a few micrometers or less is needed.
In order to overcome the problems in the prior art, it was suggested, in U.S. Pat. No. 6,127,184 to Wardlaw, to design a cuvette-like optical sample container for the cell suspension that has different optical pathlengths in different areas. In at least one area, the thickness of the liquid layer of un-diluted blood is so thin (2 to 7 microns) that monolayers of isolated RBCs are formed. In another region, the liquid layer is thicker (7 to 40 microns), and typical chain-like aggregates of RBCs (xe2x80x9cRoleauxxe2x80x9d) are forming. The thick area is used to determine the HCT, and the thin area is used to determine the volume of single red blood cells (RCV). As in the prior art, the blood plasma is stained with a fluorescent dye that is not penetrating into the RBCs.
In a method and apparatus described in U.S. Pat. No. 6,127,184, the HCT of the whole blood sample is determined according to the equation                     HCT        =                              [                          1              -                                                B                  a                                                  B                  t                                                      ]                    *          100          ⁢          %                                    (        1        )            
In equation (1), Bt is the fluorescence intensity emerging from an area of known size within a cell-free plasma region. Ba is the fluorescence intensity emerging from another area of same size, but from a region comprising RBCs in Roleaux formation. In practice, Bt is determined by measuring the fluorescence intensity in certain cell-free regions and by extrapolating to a larger size. Interestingly, no height measurement is required for the cuvette in order to determine the HCT.
The volume of single Red Blood Cells, RCV, can be determined using the equation                     RCV        =                              [                          1              -                                                B                  RBC                                                  B                  P                                                      ]                    *          A          *          d                                    (        2        )            
where, similar to equation (1), BP is the fluorescence intensity emerging from an area of known size, A, within a cell-free plasma region. BRBC is the fluorescence intensity emerging from another area of same size, but comprising a single RBC. In practice, BP is determined by measuring the fluorescence intensity in a cell-free region near a particular RBC, and by extrapolating to the full size, A. In contrast to the HCT determination, the absolute area, A, and the absolute height of the liquid layer, d, have to be known. One could also say that the absolute volume
V=A*dxe2x80x83xe2x80x83(3)
in which the single RBC is embedded, has to be known. The area, A, can be easily determined under the microscope. The determination of the height, d, near the RBC is a more complicated problem and is named xe2x80x9ccalibrationxe2x80x9d.
U.S. Pat. No. 6,127,184 discloses some methods to xe2x80x9ccalibratexe2x80x9d the optical cuvette, i.e. to determine the height, d. In one option, a square-shaped capillary of known volume is integrated into the cuvette. By measuring the fluorescence intensity reemerging from this capillary one obtains a calibration parameter C=intensity/volume. Since the fluorescence intensity per unit area is assumed to be proportional to the height of the cuvette, the height at any location can then be determined via the reemerging fluorescence intensity. In another method, U.S. Pat. No. 6,127,184 discloses the utilization of a molded calibration standard such as a well of accurately controlled depth.
However, the integration of a square-shaped capillary of known volume, or the use of a well of accurately controlled depth are likely to increase the complexity and therefore the cost of the cuvette-like optical sample container.
Consequently, there still exists a need for a calibration method for sample analyzer disposables that would not require any integrated or molded calibration tools of high precision.
It is an objective of the present invention to provide a method for calibrating a sample analyzer, and preferably, a disposable sample analyzer, and in particular a calibration method for the RCV determination in blood samples that would be exact, but would not require molded calibration tools of high precision within the sample analyzer.
According to the present invention, the above objective is achieved by depositing a sample of biological fluid, and preferably, whole blood, into a chamber, such as for example, an optical cuvette having at least two areas of different thickness, whereby in a preferred embodiment the blood plasma contains a fluorescent dye that does not diffuse into the red blood cells. The sample is put into an optical scanning instrument such as a fluorescence microscope and is illuminated with excitation light so that the plasma emits fluorescence radiation, which is detected by the microscope""s imaging photodetector. The fluorescent dye is selected so that neither the excitation light nor the emitted fluorescence light are absorbed significantly by the red blood cells.
A height value in the single-cell area of the optical cuvette is then determined by performing the following process steps of:
(a) measuring fluorescence intensity values in cell-free locations within a calibration area of sufficient height;
(b) extrapolating to the integrated fluorescence intensity, Ical, for a total calibration area of size, A, that could be expected under cell-free conditions;
(c) measuring fluorescence intensity values in cell-free locations within the single-cell area;
(d) extrapolating to the integrated fluorescence intensity, Isca, for a total area of size, A, that could be expected under cell-free conditions;
(e) determining an effective height, H, by using the equation:                     H        =                              ∫                                          -                h                            /              2                                      h              /              2                                ⁢                                    E              ⁡                              (                z                )                                      *                          D              ⁡                              (                z                )                                      ⁢                          xe2x80x83                        ⁢                          ⅆ              z                                                          (        4        )            
xe2x80x83where                               E          ⁡                      (            z            )                          =                              "AutoLeftMatch"                          [                                                sin                  ⁡                                      (                                                                                            u                          ex                                                ⁡                                                  (                          z                          )                                                                    /                      4                                        )                                                                                                              u                      ex                                        ⁡                                          (                      z                      )                                                        /                  4                                            ]                        "AutoRightMatch"                    2                                    (        5        )            
xe2x80x83where                                           u            ex                    ⁡                      (            z            )                          =                                            2              *              π                                      λ              ex                                *                                    (              NA              )                        2                    *          z                                    (        6        )            
xe2x80x83where                               D          ⁡                      (            z            )                          =                              "AutoLeftMatch"                          [                                                sin                  ⁡                                      (                                                                                            u                          em                                                ⁡                                                  (                          z                          )                                                                    /                      4                                        )                                                                                                              u                      em                                        ⁡                                          (                      z                      )                                                        /                  4                                            ]                        "AutoRightMatch"                    2                                    (        7        )            
xe2x80x83where                                           u            em                    ⁡                      (            z            )                          =                                            2              *              π                                      λ              em                                *                                    (              NA              )                        2                    *          z                                    (        8        )            
xe2x80x83and wherein NA is the numerical aperture of the microscope objective lens, xcexex is the excitation center-wavelength, xcexem is the emission center-wavelength, and z is a position within the sample on an axis parallel to the microscope""s imaging optics;
(f) calculating a quantity K with the equation:                     K        =                                            I              sca                                      I              cal                                *          H                                    (        9        )            
(g) determining an integration limit, d, that is a solution of the equation:                                           ∫                                          -                d                            /              2                                      d              /              2                                ⁢                                    E              ⁡                              (                z                )                                      *                          D              ⁡                              (                z                )                                      ⁢                          xe2x80x83                        ⁢                          ⅆ              z                                      =        K                            (        10        )            
xe2x80x83whereby d is equal to the unknown sample height (which is equal to the cuvette height) in the single-cell area that is to be determined.